{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,27]],"date-time":"2026-01-27T01:50:52Z","timestamp":1769478652144,"version":"3.49.0"},"reference-count":33,"publisher":"FapUNIFESP (SciELO)","license":[{"start":{"date-parts":[[2025,1,1]],"date-time":"2025-01-01T00:00:00Z","timestamp":1735689600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,1,1]],"date-time":"2025-01-01T00:00:00Z","timestamp":1735689600000},"content-version":"am","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,1,1]],"date-time":"2025-01-01T00:00:00Z","timestamp":1735689600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006749","name":"Burapha University","doi-asserted-by":"crossref","award":["171\/2561"],"award-info":[{"award-number":["171\/2561"]}],"id":[{"id":"10.13039\/501100006749","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Braz. J. Biol."],"abstract":"<jats:p>Abstract Quantitative real-time PCR (qRT-PCR) is a widely used method for measuring gene expression, but its reliability depends on using stable reference genes for normalization. This study assessed the expression stability of four commonly used housekeeping genes\u201418S rRNA, ef1\u03b1, rpl8, and \u03b2-actin\u2014in Amphiprion ocellaris, a socially regulated protandrous hermaphrodite. Brain and gonadal tissues were sampled from individuals at different developmental stages and sex types, including juveniles, transitional individuals, and paired breeders. To further evaluate tissue-wide stability, 11 different tissues were also analyzed in adult breeders. Gene expression stability was evaluated using RefFinder, which integrates four established algorithms. Among the candidates, 18S rRNA was consistently ranked as the most stable across tissues and analysis methods, while \u03b2-actin showed the highest variability and was the least reliable. Validation using two sex differentiation genes, cyp19a1a and cyp19a1b, normalized with 18S rRNA, confirmed biologically consistent and tissue-specific expression patterns. These findings identify 18S rRNA as the most suitable reference gene for qRT-PCR studies in A. ocellaris, with ef1\u03b1 and rpl8 as potential alternatives in gonadal tissues. Using validated reference genes is essential for accurate gene expression analysis in studies of sex differentiation and reproductive plasticity in sequentially hermaphroditic teleosts.<\/jats:p>","DOI":"10.1590\/1519-6984.297690","type":"journal-article","created":{"date-parts":[[2026,1,26]],"date-time":"2026-01-26T15:12:29Z","timestamp":1769440349000},"source":"Crossref","is-referenced-by-count":0,"title":["Tissue-specific validation of reference genes for accurate qRT-PCR analysis during sex differentiation in Amphiprion ocellaris"],"prefix":"10.1590","volume":"85","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8706-225X","authenticated-orcid":false,"given":"C.","family":"Boonphakdee","sequence":"first","affiliation":[{"name":"Burapha University,  Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-5281-9464","authenticated-orcid":false,"given":"J.","family":"Thamnawasolos","sequence":"additional","affiliation":[{"name":"Rajabhat Rajanagarindra University,  Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1560-2739","authenticated-orcid":false,"given":"T.","family":"Boonphakdee","sequence":"additional","affiliation":[{"name":"Burapha University,  Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-7861-9994","authenticated-orcid":false,"given":"T.","family":"Mataroepayotorn","sequence":"additional","affiliation":[{"name":"Burapha University,  Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"530","published-online":{"date-parts":[[2025]]},"reference":[{"issue":"15","key":"ref1","doi-asserted-by":"crossref","first-page":"5245","DOI":"10.1158\/0008-5472.CAN-04-0496","article-title":"Normalization of real-time quantitative reverse transcription\u2013PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets","volume":"64","author":"ANDERSEN C.L.","year":"2004","journal-title":"Cancer Research"},{"issue":"6","key":"ref2","first-page":"652","article-title":"18S rRNA, a potential reference gene in the qRT-PCR measurement of bisphenol A contamination in green mussels (Perna viridis) collected from the Gulf of Thailand","volume":"53","author":"BOONPHAKDEE C.","year":"2019","journal-title":"Agriculture and Natural Resources"},{"issue":"6945","key":"ref3","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1038\/424145a","article-title":"Social hierarchies: size and growth modification in clownfish","volume":"424","author":"BUSTON P.","year":"2003","journal-title":"Nature"},{"issue":"4","key":"ref4","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1373\/clinchem.2008.112797","article-title":"The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments","volume":"55","author":"BUSTIN S.A.","year":"2009","journal-title":"Clinical Chemistry"},{"issue":"1","key":"ref5","doi-asserted-by":"crossref","first-page":"13323","DOI":"10.1038\/s41598-024-63335-x","article-title":"Systematic selection of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Lutjanus erythropterus.","volume":"14","author":"CHEN L.","year":"2024","journal-title":"Scientific Reports"},{"issue":"2","key":"ref6","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1007\/s10126-010-9277-z","article-title":"Normalizing RT-qPCR data: are we getting the right answers? 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